Rapid antibiotic susceptibility testing by tracking sub-micron scale motion of single bacterial cells

ABSTRACT

A method for rapid antibiotic susceptibility testing by tracking sub-micron scale motion of single bacterial cells including obtaining a biological sample from a subject including live bacteria. Different doses of antibiotic are added to a multi-well glass slide and adding portions of the biological sample to the wells. Bacterial cells are tethered onto the surface. The tethered bacterial cells are imaged and tracked. Bacterial sub-micron motion of tethered cells is measured at the different doses. A processor performs statistical analysis on a population of cells for each antibiotic dose to generate an antibiotic dose curve proportional to the motion changes, where the antibiotic dose curve plots data including a decrease in movement over time indicating a proportional effectiveness of an antibiotic applied to a well.

TECHNICAL FIELD

The present invention relates to antibiotic susceptibility testing(AST), and, more particularly, to a method and apparatus for rapidantibiotic susceptibility testing by tracking sub-micron scale motion ofsingle bacterial cells.

BACKGROUND

Bacterial infections are a global disease burden, causing 3 millionpatient deaths and 30 million patient hospitalizations annually.¹Effective treatment of bacterial infections requires prescription ofappropriate antibiotics at the disease onset.² This requirement isespecially critical for patients with sepsis or experiencing septicshock, where mortality increases 7.6% for every hour of delayedantibiotic treatment.³⁻⁵ Antibiotic susceptibility testing (AST) is usedto identify antibiotic resistant bacterial strains and to enabletreatment with appropriate antibiotics.^(2,8) However, current ASTtechnologies take 1-3 days due to their dependence on bacterialculturing.⁷⁸ Without rapid AST, physicians often prescribebroad-spectrum antibiotics,^(9,10) which has contributed to theacceleration of bacterial antibiotic resistance.^(4,9) A faster ASTtechnique will empower physicians to prescribe, preferably within 1-2hours or less, effective narrow-spectrum antibiotics.^(11,12)

Emerging AST technologies based on detecting bacterial growth rate viameasuring cell numbers¹³⁻¹⁵, cell size^(18,17), and molecular orbiochemical markers (RNA,^(18,19) DNA,²⁰ or redox molecules²¹⁻²⁴) arebeing developed. Several culture-free, metabolism-based ASTtechnologies, which use bacterial nano-motion,²⁵⁻²⁷heat-signatures,^(28,29) and biochemical profiles,³⁰ have also beenpursued. While these technologies offer potential solutions, a rapid(e.g., 2 hours) and robust AST technology requires further development.⁸

Hydrodynamic^(31,32) and motility-induced long-range motions^(31,33) ofalive bacterial cells in the range of several microns near a surfacehave been studied using particle image velocimetry algorithms³⁴ anddigital holographic imaging technologies.³² However, short-range motion(a few nm) of surface-attached bacterial cells and their correlation tobacterial metabolism have only been recently studied using highlysensitive tools, such as atomic force microscopy^(26,36) and plasmonicimaging and tracking (PIT).^(26,36)

BRIEF SUMMARY OF THE DISCLOSURE

This summary is provided to introduce, in a simplified form, a selectionof concepts that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

A method for rapid antibiotic susceptibility testing by trackingsub-micron scale motion of single bacterial cells including obtaining abiological sample from a subject including live bacteria is disclosed.Different doses of antibiotic are added to a multi-well glass slide andadding portions of the biological sample to the wells. Bacterial cellsare tethered onto the glass surface. The tethered bacterial cells areimaged and tracked. Bacterial sub-micron motion of tethered cells ismeasured at the different doses. A processor performs statisticalanalysis on a population of cells for each antibiotic dose to generatean antibiotic dose curve proportional to the motion changes, where theantibiotic dose curve plots data including a decrease in movement overtime indicating a proportional effectiveness of an antibiotic applied toa well.

Motility-induced motion of bacterial cells in solution has beenpreviously studied using various techniques.^(32,48) Bacterial cellshave also been studied by attaching them firmly to a surface to enableclear optical imaging of the cells.⁴⁵ However, none of these approacheshave quantified the sub-μm motion of surface-tethered bacterial cellsand applied it for AST. A key enabling step in the present work is theoptimization of the antibody and APTES surface chemistry to achieveloosely tethered bacterial cells for continuous imaging and tracking ofthe sub-micron motion of each cell.

Demonstrated herein is a method for rapid (within 2 h) AST ofclinically-relevant bacteria, E. coli O157:H7³⁷ and UPEC, by quantifyingthe sub-μm motion of single cells. Compared to AFM, which measurescantilever deflections due to bacterial nano-motion in z direction, themethod disclosed herein can simultaneously image multiple bacterialcells. Compared to a previously described PIT system,²⁶ the presentmethod is simpler in both instrumentation and sensor chip preparation.The present work has focused on E. coli O157:H7³⁷ and UPEC with thepolymyxin B antibiotic. Future work will include other bacteria,including Staphylococcus aureus, ²⁵⁻²⁷ to demonstrate the broadapplication of the present method.

The sub-μm motion of surface-tethered bacterial cells is measured usinga simple bright-field imaging setup together with automated imageprocessing algorithms and determine the correlation of the sub-μm motionwith bacterial viability and metabolism. The method is applied toclinically relevant bacteria, Escherichia coli O157:H7³⁷ anduropathogenic E. coli (UPEC), and the antibiotic, polymyxin B.³⁸ UPECaccounts for ˜75% of all urinary tract infections (UTIs) and affectsover 10 million people worldwide.³⁸ Polymyxin B is a cationicpolypeptide antibiotic closely related to colistin (polymyxin E), whichis an important last-line antibiotic.³⁹ Colistin resistance has recentlybeen reported in bacterial strains that cause UTIs inpatients.^(40,41,42)

To demonstrate rapid AST, a coupling chemistry was developed to tetherbacterial cells loosely onto the glass surface and imaging processingalgorithms to quantify the sub-μm motion of tethered cells. Theantibiotic-induced sub-micron motion changes of individual cells werefurther measured, antibiotic dose dependency of the motion changes wasstudied, clinically relevant minimum bactericidal concentration of theantibiotic was determined, and rapid AST performed on human urinesamples spiked with UPEC cells.

BRIEF DESCRIPTION OF THE DRAWINGS

While the novel features of certain embodiments of the invention are setforth with particularity in the appended claims, the invention, both asto organization and content, will be better understood and appreciated,along with other objects and features thereof, from the followingdetailed description taken in conjunction with the drawings, in which:

FIG. 1 schematically shows an example of an experimental setup to imageand track bacterial cell sub-μm motions.

FIG. 2A shows an image of an E. coli O157:H7 bacterial cell withsuperimposed motion of the cell center over 20 sec shown as darkeneddots.

FIG. 2A′ shows a more detailed plot of the displacement of the cellcenter of FIG. 2A over 20 secs, reflecting the sub-μm motion of thecell.

FIG. 2B shows a plot of distance, defined as the root-mean-square of thedisplacement of cell center over 20 secs, of nine different bacterialcells. The dashed line represents average distance (D_(AVG)) of the ninebacterial cells.

FIG. 3A shows images of a bacterial cell and displacement of the cellcenter in 1×PBS before (0 min) and after polymyxin B addition (2 and 5min).

FIG. 3B shows displacement plots of the cell center recorded over 20sec.

FIG. 3C shows distances of the cell motion at 0, 2, and 5 min.

FIG. 3D shows average distance (D_(AVG)) of nine cells before and 10 minafter the addition of polymyxin B (0.5 mg/ml).

FIG. 4A-FIG. 4F illustrate plots of D_(AVG) of a population of bacterialcells in different wells at different time points. Cells weresequentially exposed to 1×PBS (bar with vertical striping, baselinemeasurement), different clinically-relevant doses of polymyxin B (barswith horizontal striping, antibiotics added immediately after thebaseline measurement), and a lethal dose of antibiotic (bars withdiagonal striping, 0.5 mg/ml of polymyxin B added after the 75-minmeasurement).

FIG. 5A-5F illustrate plots of D_(AVG) of UPEC cells in human urineexposed to increasing concentrations of polymyxin B.

FIG. 6A-FIG. 6D show an example image processing of bacterial cells toquantitate X and Y displacement and movement.

FIG. 7A-FIG. 7D show an example of motion (X and Y displacement) of alive bacterial cell compared to a fixed marker spot.

FIG. 8A-FIG. 8E show examples of dose dependency of a total number ofUPEC cells in the images.

FIG. 9A-FIG. 9C illustrate changes in sub-μm motion of a replicatingUPEC cell partially tethered on the surface.

FIG. 10A-FIG. 10C illustrate changes in sub-μm motion of a replicatingUPEC cell partially tethered on the surface.

FIG. 11A-FIG. 11C illustrate a decrease in sub-μm motion ofsurface-tethered UPEC cells exposed to 0.25 μg/ml polymyxin B.

FIG. 12A illustrates images of an increase in sub-μm motion of asurface-tethered UPEC cell exposed to 0.25 μg/ml polymyxin B over time.

FIG. 12B plots displacement distance of the cell of FIG. 12A atdifferent time points.

FIG. 13A illustrates images of cells showing a decrease in sub-μm motionof a surface-tethered UPEC cell exposed to 2 μg/ml polymyxin B overtime.

FIG. 13B plots displacement distance of the cell of FIG. 13A atdifferent time points.

FIG. 14A illustrates images of cells showing a decrease in sub-μm motionof a surface-tethered UPEC cell exposed to 2 μg/ml polymyxin B overtime.

FIG. 14B plots displacement distance of the cell of FIG. 14A atdifferent time points.

FIG. 15 shows a plot measuring inhibition of UPEC in Mueller Hintonbroth (MHB) after incubation with increasing polymyxin B concentrations.

FIG. 16 shows a table illustrating growth inhibition and bactericidalactivity of the antibiotic polymyxin B.

FIG. 17A-FIG. 17F are plots of bactericidal activity of polymyxin Bagainst UPEC in spiked urine samples.

FIG. 18A shows motion of a living bacterial cell over time is shown.

FIG. 19A and FIG. 19B show motion of a dead bacterial cell over time.

FIG. 20A-FIG. 20C show motion changes of loosely and completely tetheredbacterial cells.

In the drawings, identical reference numbers identify similar elementsor components. The sizes and relative positions of elements in thedrawings are not necessarily drawn to scale. For example, the shapes ofvarious elements and angles are not drawn to scale, and some of theseelements are arbitrarily enlarged and positioned to improve drawinglegibility. Further, the particular shapes of the elements as drawn, arenot intended to convey any information regarding the actual shape of theparticular elements, and have been solely selected for ease ofrecognition in the drawings.

DETAILED DESCRIPTION

The following disclosure describes a device for antibioticsusceptibility testing (AST). Several features of methods and systems inaccordance with example embodiments are set forth and described in thefigures. It will be appreciated that methods and systems in accordancewith other example embodiments can include additional procedures orfeatures different than those shown in the figures. Example embodimentsare described herein with respect to a rapid AST apparatus and methodfor measuring the sub-micron motion of surface-tethered bacterial cells.However, it will be understood that these examples are for the purposeof illustrating the principles, and that the invention is not solimited.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense that is as “including, but not limited to.”

Reference throughout this specification to “one example” or “an exampleembodiment,” “one embodiment,” “an embodiment” or combinations and/orvariations of these terms means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present disclosure. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

Definitions

Generally, as used herein, the following terms have the followingmeanings when used within the context of microarray technology:

The articles “a” or “an” and the phrase “at least one” as used hereinrefers to one or more.

As used herein, (3-Aminopropyl)triethoxysilane (APTES) is an aminosilanefrequently used in the process of silanization, the functionalization ofsurfaces with alkoxysilane molecules.

As used herein, “AST” means antibiotic susceptibility testing of cells.

“Deep Learning,” as used herein, is used in its generally acceptedmeaning as a class of machine learning algorithms using a cascade ofmany layers of nonlinear processing units, as for example neuralnetworks and adaptive processors, that can be based on unsupervised orsupervised learning, pattern analysis applications and the like.

“Minimal Inhibitory Concentration (MIC)” is used in its generallyaccepted meaning as the lowest drug concentration that prevents visiblemicroorganism growth.

“Minimum Bactericidal Concentration (MBC)” is used in its generallyaccepted meaning as the lowest concentration of an antibacterial agentrequired to kill a particular bacterium.

As used herein, “plurality” is understood to mean more than one. Forexample, a plurality refers to at least two, three, four, five, ten, 25,50, 75, 100, 1,000, 10,000 or more.

As used in this specification, the terms “processor” and “computerprocessor” encompass a personal computer, a tablet computer, a smartphone, a microcontroller, a microprocessor, a field programmable objectarray (FPOA), a digital signal processor (DSP), an application-specificintegrated circuit (ASIC), a field programmable gate array (FPGA), aprogrammable logic array (PLA), or any other digital processing engine,device or equivalent capable of executing software code includingrelated memory devices, transmission devices, pointing devices,input/output devices, displays and equivalents.

“Obtaining” is understood herein as manufacturing, purchasing, orotherwise coming into possession of.

Example Embodiments

Referring now to FIG. 1, an example of performed an experimental setupto image and track bacterial cell sub-μm motions is schematically shown.There shown is a multistep method of 100 including the act ofintroducing a biological sample 102, such as a urine sample, imagingtethered bacteria 104, introducing an antibiotic 106, analyzingbacterial motion at difference antibiotic doses 108 and plotting anantibiotic dose curve 110. At step 1, human urine samples 10, forexample, are spiked with live bacteria and added to a multi-well glassslide 12, thus allowing bacterial cells 5 to tether onto the glasssurface 14. At step 2, wells 15 of the slide are imaged by lightmicroscopy 120, and the motions of the tethered bacterial cells arequantitated via image analysis as indicated by a camera 122. The cameramay include or be connected to a processor for image processing andother software operations. At step 3, different doses of antibiotics areadded to the wells on the slides, and the changes in bacterial cellmotions are recorded. At step 4, image analysis reveals that thebacterial cell population decreases motion (represented by averagedistance—D_(AVG); discussed below) upon antibiotic exposure. Bars 130show motion before adding antibiotic. Bars 132 show motion afterantibiotic. Statistical analysis on a population of cells for eachantibiotic dose is performed, and an antibiotic dose curve is obtained.Plot 110 represents a plot of normalized D_(AVG) in nm plotted againstantibiotic concentration in μg/ml. Note that after 75 minutes thenormalized D_(AVG) decreases substantially from a normalized value of 1to below 0.2 nm, as the antibiotic concentration increases from 0 to 4μg/ml.

Referring now to FIG. 2A an image of an E. coli O157:H7 bacterial cellwith superimposed motion of the cell center over 20 sec shown asdarkened dots is shown. E. coli O157:H7 cells 202 tethered to the glassslide were imaged. The cell 200, exhibits motion as represented by dotsin frame 202. Scale Bar 203 is 1 μm. 43 The center of each bacterialcell was identified in each image frame (described in more detail belowwith reference to FIG. 6A-FIG. 6D) and plotted as data points 204 inFIG. 2A′.

FIG. 2A′ shows a more detailed plot of the displacement of the cellcenter of FIG. 2A over 20 secs, reflecting the sub-μm motion of thecell. As there shown, plot 202 plots motion data 204 in nm in X and Ycoordinates ranging from −400 nm to 400 nm. The center positions of thebacterial cell (center) at different moments are shown and reveal thesub-μm scale motion of the cell. For quantitative analysis of the sub-μmmotion of the bacterial cell, “Distance” is defined as theroot-mean-square of the position over 20 seconds. Note that the motionranges from about −200 nm to 200 nm displacement along both coordinates.

FIG. 2B shows a plot of distance, defined as the root-mean-square of thedisplacement of cell center over 20 secs, of nine different bacterialcells. The dashed line 210 represents average distance (D_(AVG)) of thenine bacterial cells. The distances for different bacterial cells variedfrom 75 to 842 nm, with an average distance (D_(AVG)) of 265 nm. Forquantitative analysis of the sub-μm motion of the bacterial cell, wedefined “Distance” as the root-mean-square of the position over 20seconds. For an individual bacterial cell, the distance varied with timeover triplicate 20 s recordings and reached its peak within 5 seconds Bytracking the position of a marker (pillars attached inside amicrofluidic cassette) in the image, the measured distance error was −25nm (described below with reference to FIG. 7A-FIG. 7B). That is muchsmaller than the average distance (278 nm) of a typical bacterial cellshown in FIG. 2A. These results demonstrated that the bacteria cellstethered on the surface experienced sub-μm motion, and the imagingalgorithm accurately tracked the motion.

Referring now to FIG. 3A, images of a bacterial cell and displacement ofthe cell center in 1×PBS before (0 min) and after polymyxin B addition(2 and 5 min) are shown. Images 301, 302, 303 of a bacterial cell anddisplacement of the cell center 310 in 1×PBS before (0 min) and afterpolymyxin B addition (2 and 5 min). Antibiotic effects on the sub-μmmotion were examined using a lethal dose of polymyxin B (0.5 mg/ml).Images 301, 302, 303 show the bright-field images of an alive bacterialcell before, 2 min, and 5 min, respectively after exposure to 0.5 mg/mlpolymyxin B, where the positions of the bacterial cell before and afterthe exposure to polymyxin B, represented by indicia 310, 310A and 310Brespectively. Scale bar 311 is set at 1 μm. #, p=0.065.

FIG. 3B shows displacement plots of the cell center recorded over 20sec. The corresponding sub-μm motion and positions of the bacterial cellare more clearly shown in plots 320,322,324.

FIG. 3C shows distances of the cell motion at 0, 2, and 5 min. Theaverage E. coli O157:H7 distance over 20 sec. in 1×PBS was 278 nm asindicated by bar 350, which reduced to 225 nm within 2 min afteraddition of 0.5 mg/ml polymyxin B, indicated by bar 352 and further to65 nm at 5 min post-antibiotic exposure as plotted in plot 324 andrepresented by bar 354. The remaining motion (65 nm) indicated that thebacteria had ceased activity since this amount of motion was likely dueto the Brownian motion as a fixed object in the image had a motion of 25nm (described below with reference to FIG. 7A-FIG. 7B). To ensurecomplete killing of the bacteria after 5 min exposure to the high,lethal dose of polymyxin B, overnight culturing of the cells yielded noviable cells. These results indicated that the sub-μm motion in therange of 50-65 nm is a good signature of a bacterial metabolic state.

Extrapolating further information from the results of FIG. 3C, inoperation, a processor can be operated to perform statistical analysison a population of cells for each antibiotic dose to generate anantibiotic dose curve proportional to the motion changes as describedabove. Further, using an imaging device and a processor, a distancerange of bacterial cell motion can be determined as shown in the rangeof about 50 nm to about 65 nm (likely due to the Brownian motion as afixed object in the image had a motion of 25 nm). Then, using aprocessor, the dose for which a distance range of bacterial cell motionis between 50 nm and 65 nm can be determined at a predetermined time ofexposure, as for example, five minutes or more. This restriction inrange of motion signals that the corresponding dose comprises the MBC.

The displacement associated with the Brownian motion of a freely movingbacterial cell in solution is given by (2Dt)^(1/2) according to thediffusion model, 45 where D, the diffusion coefficient of bacteria, is˜10-5 cm²/s and t is the time scale. For a time scale of 5-20 sec, thisBrownian motion is several microns for free moving cell, much greaterthan the observed 65 nm for our cells attached to the surface. Thisdiscrepancy arises because the bacterial cells are attached to thesurface in our case and their motion depends upon surface interactions.

While the loosely tethered cells showed a large decrease in the sub-μmmotion upon antibiotic exposure, tightly bound cells displayed smallermotion changes (data not shown). A robust AST method must work for abacterial population that contains a mixture of loosely and tightlybound cells. To demonstrate this capability, antibiotic effects on the“average distance” (D_(AVG)) were studied for a population of bacterialcells that included both tightly bound and partially bound cells. Forthis mixed population of bound cells, the D_(AVG) was 265 nm (FIG. 3C)before the addition of polymyxin B and dropped to 123 nm at 10 minpost-antibiotic exposure (FIG. 3D).

Referring now to FIG. 3D, average distance (D_(AVG)) of nine cellsbefore and 10 min after the addition of polymyxin B (0.5 mg/ml) isshown. Plot 347 shows histogram bar 360 representing motion at time 0.Histogram bar 362 represents time at 10 minutes after introduction ofthe polymyxin B. Note that the displacement between time zero and timepeople to 10 minutes is substantially reduced from about 260 nm to about110 nm.

Referring now jointly to FIG. 4A-FIG. 4E, D_(AVG) of a population ofbacterial cells in different wells at different time points isgraphically shown. Cells were sequentially exposed to 1×PBS, differentclinically-relevant doses of polymyxin B, and a lethal dose ofantibiotic (0.5 mg/ml of polymyxin B added after the 75-minmeasurement). D_(AVG) of cells in wells with 0 (FIG. 4A), 0.25 (FIG.4B), 0.5 (FIG. 4C), 2 (FIG. 4D), and 4 μg/ml of polymyxin B (FIG. 4E).

To apply the above motion-tracking method to AST of the clinicallyrelevant UPEC strain, UPEC cells were tethered to the glass surface viaAPTES surface chemistry and conjugation to the amine group of abacterial surface protein. Antibiotic dose-dependent experiments wereperformed in a multiplexed format using multi-well slides and a 40×objective. First, baseline images were captured at 0 min for each well.Subsequently, polymyxin B at clinically relevant concentrations (4, 2,0.5, and 0.25 μg/ml) was added to different wells, and images wererecorded at 15 min intervals for 75 min. Finally, as a positive control,a bactericidal dose (0.5 mg/ml) of polymyxin B was added to all wells,and images were recorded after incubation for 15 min (at 90 min). Thecontrol well (Well 1) initially harbored 100 tethered cells (asdescribed below with reference to FIG. 8A) with an initial D_(AVG) of228 nm (FIG. 4A). Over time (0-75 min), the number of cells on thesurface increased gradually (as described below with reference to FIG.8A) due to replication of the surface-tethered cells (as described belowwith respect to FIG. 9A-10B). During the first 15 min, D_(AVG) increasedfrom 228 nm to 388 nm, followed by a gradual decrease from 388 to 227 nmover 75 min (FIG. 4A). This decrease in D_(AVG) in the absence ofantibiotics may be attributed to the depletion of nutrients oradaptation of bacteria to the environment.46 In the presence of lethalpolymyxin B concentration, D_(AVG) decreased significantly from 227 nmto 64 nm in 75 min (FIG. 4A; p<0.05). D_(AVG) vs. time was analyzed forpolymyxin B varying between 0.5-4 μg/ml (FIG. 4B-FIG. 4E), showing clearcorrelation between the sub-μm motion and antibiotic concentration.Within this concentration range, the number of cells remained constantover 75 min (as described below with reference to FIG. 8A-FIG. 8E),indicating growth inhibition. At higher clinically relevant doses (e.g.2 and 4 μg/mL), the D_(AVG) value dropped post antibiotic addition,which shows the clear correlation between the sub-μm motion andantibiotic action. These findings validated that the sub-μm motion ofsurface-tethered UPEC cells with the APTES chemistry is correlated withthe bacterial metabolic activity, thus enabling use for AST.

While the results described above are population-based analyses, thistechnology also allows for single cell analysis to provide fundamentalphenotypic information for individual cells. For example, cells in thecontrol well (without antibiotics) exhibited increased motionimmediately after replication, followed by decreased motion after addinga lethal dose of polymyxin B (as described in more detail below withrespect to FIG. 9A-10B). Another example is the observation of twophenotypic subpopulations detected upon exposure to low dose (0.25μg/ml) polymyxin B. One subpopulation showed decreased motion (FIG. 4B)(as described in more detail below with respect to FIG. 11A-FIG. 11C),while the second subpopulation continued to replicate and showedincreased motion with 0.25 μg/ml polymyxin B (as described in moredetail below with respect to FIG. 12A-FIG. 12C). While analysis ofdifferent cells revealed large heterogeneity in cellular motionresponses to low dose polymyxin B, at higher lethal polymyxin Bconcentration, all bacterial cells decreased motion (as described inmore detail below with respect to FIG. 11A-FIG. 14B). These resultsshowed that different cells in a sample may display different phenotypicand resistance responses for an antibiotic dose and that this singlecell analysis capability allows detection of subpopulations of resistantbacteria in a sample.

Referring to FIG. 4F, an antibiotic dose curve of normalized D_(AVG) at75 min. is shown, where p<0.05; ns, means not significant. To obtain anantibiotic dose-response curve, the 75-min D_(AVG) values werenormalized between the 0-min D_(AVG) values and the 90-min D_(AVG)values as well as plotted against polymyxin B concentration, thusrevealing decreased D_(AVG) with increasing polymyxin B concentrations.At 4 μg/ml of polymyxin B, D_(AVG) was similar to the D_(AVG) of thelethal concentration (0.5 mg/ml) polymyxin B, indicating that 4 μg/mlpolymyxin B is the minimum bactericidal concentration (MBC) at 75 min(referred to as MBC75 min). The polymyxin B minimum inhibitoryconcentration (MIC) was determined using the standard microdilutionbroth assay, thus revealing an MIC value of 1 μg/ml after 16 hincubation (as described in more detail below with respect to FIG. 15).Subsequent quantitative plating determined the standard incubation (16h) MBC of 2 μg/ml (as described in more detail below with respect toFIG. 16). The MBC75 min value determined by this rapid AST method islower than the MBC measured by the gold standard culturing method, butit is within the QC range of Clinical & Laboratory Standards Institute(CLSI), indicating that the present method can provideclinically-significant MBC values and perform rapid AST.

Referring now to FIG. 5A-5F, plots of D_(AVG) of UPEC cells in humanurine exposed to increasing concentrations of polymyxin B are thereillustrated. UPEC cells (5×106 cfu/ml) were added to human urinesamples, and D_(AVG) was determined over time in the absence andpresence of clinically-relevant concentrations of polymyxin B. The barswith vertical striping represent the baseline D_(AVG) measurement ofUPEC cells in urine. D_(AVG) measurement of UPEC cells upon exposure toclinically-relevant concentrations of polymyxin B (bars with horizontalstriping; antibiotics added immediately after the baseline measurement)and a high, bactericidal concentration of polymyxin B (bars withdiagonal striping, 0.5 mg/ml added after the 90-min measurement).D_(AVG) of cells in wells with (a) 0, (b) 0.25, (c) 1, (d) 2, (e) 4, and(f) 8 μg/ml of polymyxin B. *, p<0.05; ns, not significant.

To test the feasibility of an example of a method for analyzingclinically relevant samples, urine samples collected from healthypatients were spiked with UPEC bacterial cells. Following surfacetethering, images were recorded from six different wells with polymyxinB doses varying from 0 to 8 μg/ml. After 90 min incubation with 0.25, 1,or 2 μg/ml polymyxin B, D_(AVG) either remained unchanged or increasedover time (FIGS. 5b-d ) compared to the urine samples without polymyxinB, indicating that the cells are viable and exhibit movement in urinesamples with polymyxin B concentrations below 2 μg/ml (FIG. 5A). Afteradding a lethal dose (0.5 mg/ml) of polymyxin B, D_(AVG) decreasedsignificantly (p<0.05) within 15 min (FIG. 5A-5D). At clinicallyrelevant concentrations of 4 and 8 μg/ml polymyxin B, D_(AVG) decreasedsignificantly (p<0.05) to D_(AVG) values similar to the lethal (0.5mg/ml) polymyxin B dose (FIG. 5E and FIG. 5F), revealing a polymyxin BMBC90 min of 8 μg/ml against UPEC in human urine samples.

By repeating these experiments with increasing concentrations of UPECcells in urine samples, the polymyxin B MBC90 min was determined to bebetween 4 and 8 μg/ml (Table 1). These MBC data corroborated theprevious experiments with 5×106 cfu/ml and standard culturing methodswhich proceeded for 16 h (see also FIG. 17A-FIG. 17F). These resultsfurther demonstrated the ability of the instant technique to performrapid AST within 2 h for human urine samples.

TABLE 1 MBC of polymyxin B against UPEC cells in human urine samples.UPEC concentration Rapid AST MBC Culture AST MBC in urine 90 min (16 h)5 × 106 cfu/ml 8 μg/ml ND^(a) 107 cfu/ml 4 μg/ml ND^(a) 108 cfu/ml 4μg/ml 2 μg/ml ^(a)ND not determined

Examples

Materials

Lyophilized pellets of E. coli O157:H7 (ATCC 43888) were purchased fromFisher Scientific and UPEC E. coli strain CFT073 was purchased fromATCC. Human urine samples, pooled from 20 healthy patients, wereacquired from Bioreclamation IVT (Westbury, N.Y.) and stored at −80° C.(3-Aminopropyl) triethoxysilane (APTES) was purchased from Sigma-Aldrich(St. Louis, Mo.), aliquoted to smaller volumes under vacuum, and storedat 4° C. in a desiccator. Affinity-purified goat anti-E. coli O157:H7IgG polyclonal antibodies were purchased from Kirkegaard and PerryLaboratory Inc. (Gaithersburg, Md.). Stock solution of antibodies wereprepared by dissolving in 1 ml PBS (1X) and stored in aliquots at −20°C. 1-Mercapto-11-undecyl hexa (ethylene glycol) (PEG) andcarboxyl-terminated hexa(ethylene glycol) undecane thiol (PEG-COOH) werepurchased from Nanoscience Instruments (Phoenix, Ariz.). Polymyxin B waspurchased from Sigma-Aldrich, dissolved in 1×PBS at a stockconcentration of 10 mg/ml, and stored in the dark at 2-8° C. accordingto manufacturer instructions. Other reagents were purchased fromSigma-Aldrich.

Growth and Preparation of Bacteria

The lyophilized E. coli O157:H7 bacteria were suspended in PBS andcentrifuged at the speed of 50×g for 1 min to pellet the charcoal. Thesupernatant, containing bacteria, was collected and centrifuged at2000×g for 15 min to pellet the bacteria. The bacterial pellet wasresuspended in 1 ml of 1×PBS and mixed thoroughly. After 3 rounds ofpurification, the bacteria were resuspended in PBS with 5% glycerol andstored in 20 μl aliquots at −80° C. Similarly, E. coli strain CFT073strain was cultured on solid Luria agar, suspended in PBS with 5%glycerol, and frozen in aliquots at −80° C.

An aliquot of frozen E. coliO157:H7 or E. coli CFT073 strain was thawedand used to inoculate 3 ml of Luria broth (LB) one day before theexperiments. The overnight, saturated culture grown at 37° C. wasdiluted into fresh LB at a concentration of ˜10⁷ cfu/ml and grown at 37°C. with gentle rotary mixing until the cultures reached an OD₆₀₀ of0.56, indicating the mid-logarithmic phase of growth. The correspondingconcentration of the bacteria was 4.67×10⁸ cfu/ml. Bacterial cells werecollected by centrifugation at 2000×g for 15 min and resuspended in 1 mlPBS (1X) to an OD of 0.56. For urine experiments, pooled urine sampleswere sterilized via passage through a 0.2 μm filter and inoculated withfreshly cultured UPEC cells to the desired concentration.

Sensor Chip Surface Functionalization

Clean BK7 glass coverslips were coated with 1.5 nm chromium and 48 nmgold and used as sensing chips. The chips were rinsed with deionizedwater and ethanol multiple times followed by drying with nitrogen gasand cleaning with a hydrogen flame. For antibody surface, the cleanedchips were submerged in 1 mM PEG/PEG-COOH ethanol solution and left inthe dark for 24 h to coat a PEG/PEG-000H self-assembled monolayer (SAM)on each chip. The PEG/PEG-000H SAM-coated chips were activated with 500μl of a freshly prepared mixture of 0.1 M NHS and 0.4M EDC in 1:1 ratioto produce NHS ester receptors, which react with the primary aminegroups on the antibodies via an amide bond. Chips with activatedPEG/PEG-COOH SAM were cleaned with deionized water and blown dry withnitrogen gas. Polyclonal anti-E. coli O157:H7 IgG antibodies dissolvedin 20 mM sodium acetate, pH 5.5 at a concentration of 30 μg/ml wereimmediately applied to the NHS/EDC-activated surfaces and incubated for30 min. The antibody-coated chips were again cleaned with deionizedwater and dried with nitrogen gas prior to bacterial cell capture andimaging.

For the APTES surface, 22×60 mm BK7 glass slides from VWR (Radnor, Pa.)were used. The glass slides were thoroughly cleaned with deionized waterand ethanol and dried with nitrogen gas. The glass slide was activatedwith freshly prepared 1% APTES in 95% ethanol for 15 sec. to attach theAPTES linker to the sensor surface. The APTES linked sensor chips wereagain cleaned with ethanol and dried with nitrogen gas prior tobacterial cell capture on the imaging setup. A black permanent markerspot was placed beneath the coated surface of the glass slide foralignment purposes. A reusable, self-adhering multi-well FlexiPERM(Sarstedt) attachment was affixed to the top of the slide.

Bacterial Immobilization

E. coli O157:H7 cells (20 μI) were added to antibody-coated sensor chipscontaining 500 μl PBS (1X). Cells were tethered onto the sensor surfaceafter 10-15 min incubation at room temperature. Chips were washed withPBS buffer to remove untethered bacterial cells. During incubation in LBat room temperature, tethered bacterial cells were observed elongating,indicating that the tethered cells were viable and metabolically active.

UPEC cells suspended in urine or 1×PBS were added to the APTES coatedslides with attached FlexiPERM multiwells. Cells were tethered to thesurface after 10-15 min incubation at room temperature, and unattachedbacterial cells were removed by washing the chips with PBS.

Drug Perfusion System

A gravity-based multichannel drug perfusion system (Warner Instrument,Hamden, Conn.) was used to deliver medium and buffers to sensor chipwells. Sample solutions flowed at a rate of 330 μI/min with thetransition time between different solutions in the range of 1-2 sec. Theflow system was stopped and stabilized for 5 min before recordingvideos. Antibiotics were manually pipetted into the wells for all UPECexperiments.

Imaging Setup

The imaging setup consisted of an inverted microscope (Olympus IX-70)(FIG. 1).⁵⁰⁻⁵² A 60× oil immersion objective with a numerical aperture(NA) of 1.49 or a 40× objective (NA 0.75) was used to perform theexperiments. The glass slides were placed on a motorized microscopestage (BioPrecision2 X-Y Stage, Ludl Electronic Products Ltd.,Hawthorne, N.Y.) above the objective lens. A top mounted white lightsource was used to illuminate the sample and a CCD (Pike-032B, AlliedVision Technologies, Newburyport, Mass.) or a CMOS camera(GS3-U3-23S6M-C, Point Grey Research, Richmond, BC, Canada) was used torecord images. The assembled glass chip with an attached FlexiPERM wellwas then mounted onto the microscope stage. Light intensity was adjustedto obtain the best image contrast without image saturation.

Image Collection and Processing

Images were recorded and converted into binary images using segmentationalgorithms previously developed⁵³ and described in further detail belowwith respect to FIG. 6A-FIG. 6D.

Broth Microdilution Assay

Broth microdilution assay was used to determine the polymyxin B MIC andMBC following a standard protocol.⁵⁴ Exponential phase UPEC CFT073cultures were grown as described above, and bacterial suspensions (1×10⁸cfu/ml) were prepared in Mueller Hinton broth. Bacterial suspensions(100 μl of 5×10⁶, 10⁷, or 10⁸ cfu/ml) were added to wells of 96-wellmicrotiter plates containing polymyxin B (0.125-8 μg/ml). The MIC wasdetermined by measuring the absorbance at 600 nm after 16 h standingincubation at 37° C. Cell viability and MBC values were determined byplating duplicate 10-fold serial dilutions for each sample onto LuriaBroth agar plates and enumerating colonies after 16 h incubation at 37°C. The MBC value was determined as the minimum antibiotic concentrationthat failed to yield any positive bacterial cultures.

Statistical Analysis

Paired student t-tests were used to analyze statistical differencesbetween different values (see FIG. 3A-FIG. 3D). Repeated measuresone-way ANOVA and Games-Howell post hoc test were used to assessstatistical significance between different time points (see FIG. 4A-FIG.5F). Custom MATLAB scripts were generated to perform statisticalanalysis.

Detailed Image Collection and Processing Steps for Quantification ofBacterial Cells Displacement Referring now to FIG. 6A-FIG. 6D, anexample image processing of bacterial cells to quantitate X and Ydisplacement and movement is shown. FIG. 6A shows an example of an image(greyscale image) of a bacterial cell captured using the imaging setup.FIG. 6B shows an example where the greyscale image of a bacterial cellis converted into a binary image with the superimposed center of thecell 603. FIG. 6C shows an example where the center of the cell isplotted over 20 sec. of video to obtain the displacement, and thusmovement, of the cell. Scale bar 601 is 1 μm.

All E. coli O157:H7 experiment image sequences were collected at 106 fpsat a pixel resolution of 640×480 using the Pike-032B CCD camera. Alluropathogenic E. coli (UPEC) experiment image sequences were collectedat 26.6 fps at a pixel resolution of 1920×1200 via the PointGrey CMOScamera. The stage was translated to each well sequentially at the markerspot area and recorded 5 sec image sequences for each well. This processwas repeated every 15 min across multiple time points. The microscopefocus was set to image bright bacterial cells with darker backgrounds inthe greyscale mode. All images were processed using MATLAB programs andImageJ scripts.

Greyscale images were converted into binary images using a MATLABscript. In the greyscale images, the focus of the microscope has beenadjusted so that the bacterial cells were brighter (higher pixelintensities) compared to the background region. For every greyscaleimage (FIG. 6A), a unimodal histogram of pixel intensities was plotted(FIG. 6B). From the histogram of pixel intensities (FIG. 6B), athreshold (T) value was then determined at the point of drop in theintensity histogram towards the higher pixel intensity tail (right sidein FIG. 6B). All pixel intensities greater than the threshold value Twere converted to 1, while all the pixel intensities lower than T wereconverted to 0. The binary images segmented the bacterial cells from thebackground. Standard morphological operations were performed on thebinary images to improve the segmentations, including removing spurpoints, breaking H-connected sections, and filling holes. The greyscaleimages were thus converted into binary images with segmented bacterialcells distinct from the background (FIG. 6C).

To quantify bacterial motion, region props MATLAB command was used toobtain the “Centroid” 603 for each segmented cell in the binary images.The centroid is the X and Y coordinates of the bacterial cell center andis calculated as the mean of all non-zero pixels which the bacterialcell occupies. Next tracked the cell motion was tracked over 20 sec. forE. coliO157:H7 experiments and 5 sec. for UPEC experiments bydetermining the centroid of each segmented cell in the image sequence.To ensure proper tracking of individual cell movement, the center of thebacterium in each image was compared to the previous frame to ensurethat the movement of centroids is within the cell length. Finally, themotion of the bacteria was plotted as the X and Y displacement of thecenter (FIG. 6D). The algorithms also counted the total cells tracked asthe measure of total cells in the well.

The X and Y displacement of the cell center was calculated bysubtracting the X and Y coordinates of an individual cell from thecell's average position over the length of the image sequences. The“Distance” moved by the center of a bacterial cell was calculated byusing the formula Distance=σx²+σy², where σx and σy represents thestandard deviation of X and Y displacement, respectively. In addition,D_(AVG), the average distance for a population of cells in an imagesequence was calculated as the mean value of all individual celldistances.

Referring now to, FIG. 7A-FIG. 7D an example of motion (X and Ydisplacement) of a live bacterial cell compared to a fixed marker spotis shown. In particular, FIG. 7A shows an image of a partially tetheredbacterial cell captured using the imaging setup with superimposeddisplacement of the center shown via dots 703. Magnification andquantitation of the displacement of the cell center over 20 sec.,revealing a range of a few hundred nanometers (FIG. 7B).

FIG. 7C shows an image of a fixed marker spot captured using the imagingsetup with superimposed displacement of center shown via dots 705 (asbest shown in FIG. 7D). Magnification and quantitation of thedisplacement of the center of the marker spot over 20 sec, indicatingthat the measurement noise level is −25 nm. Scale bar 701=1 μm.

Referring now to FIG. 8A-FIG. 8E, examples of dose dependency of a totalnumber of UPEC cells in the images are shown. Total number of bacterialcells counted by an image processing algorithm in images captured frommultiple wells at different time points. Cells were sequentially exposedto 1×PBS where bars with vertical striping each represent a baselinemeasurement. Different clinically-relevant doses of polymyxin B (barswith horizontal striping, antibiotics added immediately after thebaseline measurement), and a lethal dose of antibiotic (bars withdiagonal striping, 0.5 mg/ml added after the 75 mins measurement).Polymyxin B concentrations: (a) 0, (b) 0.25, (c) 0.5, (d) 2, and (e) 4μg/ml.

Referring now to FIG. 9A-FIG. 9C, changes in sub-μm motion of areplicating UPEC cell partially tethered on the glass surface are shown.FIG. 9A shows images of a dividing UPEC bacterial cell with superimposedcenter displacement over time in 1×PBS (FIG. 9A, panels a1-a3) and afteraddition of 0.5 mg/ml polymyxin B before T=90 min (FIG. 9A, panel a4).FIG. 9B shows magnified plots (FIG. 9B, panels b1-b4) of the centerdisplacements shown in FIG. 9A panels a1-a4. FIG. 9C illustrates wheredisplacement distance showed an increase as the cell replicates (FIG. 9Apanel a3) into two daughter cells 903, 904 and a subsequent decreaseafter adding a lethal dose of polymyxin B (0.5 mg/ml) (bars withdiagonal striping). Scale bars are the same in each of the FIG. 9Apanels and are equal to 1 μm.

FIG. 10A-FIG. 10C illustrate changes in sub-μm motion of a replicatingUPEC cell partially tethered on the glass surface. Images of a dividingUPEC bacterial cell with superimposed center displacement over time in1×PBS (FIG. 10A, panels a1-a3) and after addition of 0.5 mg/ml polymyxinB over time in 1×PBS (FIG. 10A, panels a1-a3) and after addition of 0.5mg/ml polymyxin B before T=90 min (FIG. 10A, panel a4).

Referring more specifically now to FIG. 10B, there shown are magnifiedplots (FIG. 10B, panels b1-b4) of the center displacements shown in FIG.10A, panels a1-a4. Referring more specifically now to FIG. 10C, thereshown is an illustration of how displacement distance increased after 30min as the cell elongates and replicates (as shown in FIG. 10A panel a2)and subsequently decreased after one daughter cell failed to tether ontothe glass surface at 60 min. Displacement distance was further decreasedafter adding lethal a lethal dose of polymyxin B (0.5 mg/ml) (bars withdiagonal striping). As above, the scale bars shown in the lowerright-hand corner of the images equal 1 μm.

Referring now to FIG. 11A-FIG. 11C illustrate a decrease in sub-μmmotion of surface-tethered UPEC cells exposed to 0.25 μg/ml polymyxin B.FIG. 11A shows images of two individual UPEC bacterial cells withsuperimposed center displacement over time in 1×PBS (FIG. 11A, panel a1)and after addition of 0.25 μg/ml (FIG. 11A, panels a2 and a3) or 0.5mg/ml (FIG. 11A, panel a4) of polymyxin B. FIG. 11B-FIG. 11C showdisplacement distance of the UPEC cell at different time points. Thecell exhibited distance decreases at 30 and 60 min after adding 0.25μg/ml of polymyxin B (bars with horizontal striping). This cell isrepresentative of subpopulation 1, whereby low dosage of polymyxin Bdecreased the D_(AVG) of the population. The distance further decreased(bars with diagonal striping) after adding a lethal dosage (0.5 mg/ml)of polymyxin B. As above, the scale bars shown in the lower right-handcorner of the images equal 1 μm.

Referring now to, FIG. 12A images of an increase in sub-μm motion of asurface-tethered UPEC cell exposed to 0.25 μg/ml polymyxin B over timeare illustrated. Images of an individual UPEC bacterial cell withsuperimposed center over time in 1×PBS (FIG. 12A, panel a1) and afteraddition of 0.25 μg/ml (FIG. 12A, panels a2 and a3) or 0.5 mg/ml (FIG.12A, panel a4) of polymyxin B are shown. As above, the scale bars shownin the lower right-hand corner of the images equal 1 μm. FIG. 12B plotsdisplacement distance of the cell of FIG. 12A at different time points.Displacement distance of the cell as shown at different time points. Thecell showed a consistent distance increase at 30 and 60 min after adding0.25 μg/ml of polymyxin B (bars with horizontal striping). This cell isrepresentative of subpopulation 2, which exhibited motion increasesafter the addition of a low concentration of polymyxin B. Distancedecreased (bars with diagonal striping) after adding a lethal dosage(0.5 mg/ml) of polymyxin B, thus validating the metabolic origins of themotion.

FIG. 13A illustrates images of cells showing a decrease in sub-μm motionof a surface-tethered UPEC cell exposed to 2 μg/ml polymyxin B overtime. Images of a UPEC bacterial cell with superimposed motion over timein 1×PBS (FIG. 13A, panel a1) and after addition of 2 μg/ml (FIG. 13A,panels a2 and a3) or 0.5 mg/ml (FIG. 13A, panel a4) of polymyxin B. Asabove, the scale bars shown in the lower right-hand corner of the imagesequal 1 μm.

FIG. 13B plots displacement distance of the cell of FIG. 13A atdifferent time points. Displacement distance of the cell at differenttime points. Cells exhibited distance decreases at 30 and 60 min afteradding 2 μg/ml polymyxin B (bars with horizontal striping). Thedecreased distance is similar in scale to the distance after adding thebactericidal concentration of 0.5 mg/ml polymyxin B (bars with diagonalstriping), indicating cellular death after adding 2 μg/ml polymyxin B.

Referring now to FIG. 14A, images of cells showing a decrease in sub-μmmotion of a surface-tethered UPEC cell exposed to 2 μg/ml polymyxin Bover time are illustrated. Images of an individual UPEC bacterial cellwith superimposed motion over time in 1×PBS (FIG. 14A, panel a1) andafter addition of 2 μg/ml (FIG. 14A, panels a2 and a3) and 0.5 mg/mlpolymyxin B (FIG. 14A, panel a4). As above, the scale bars shown in thelower right-hand corner of the images equal 1 μm.

Referring now to FIG. 14B, displacement distance of the cell of FIG. 14Aat different time points is plotted. Cells exhibited distance decreasesat 30 and 60 min after adding 2 μg/ml polymyxin B (bars with horizontalstriping). Displacement distance of the cell at different time points.Cells exhibited distance decreases at 30 and 60 min after adding 2 μg/mlpolymyxin B (bars with horizontal striping). The distance was furtherdecreased after adding the bactericidal concentration of 0.5 mg/mlpolymyxin B (bars with diagonal striping). This result indicates thatthis individual cell is viable, but is exhibiting decreased motion afteradding 2 μg/ml polymyxin B.

Referring now to FIG. 15, a plot measuring inhibition of UPEC in MuellerHinton broth (MHB) after incubation with increasing polymyxin Bconcentrations is shown. Growth inhibition of UPEC in Mueller Hintonbroth (MHB) after incubation with increasing polymyxin B concentrations.UPEC cells (5×10⁶/ml) were incubated in MHB for 16 h at 37° C., followedby optical density measurements (OD₆₀₀). Average and standard deviationOD₆₀₀ measurements collected from triplicate experiments were plotted.

Referring now to FIG. 16, a table illustrating growth inhibition andbactericidal activity of the antibiotic polymyxin B is shown. Afterdetermining growth inhibition after

16 h incubation at 37° C., 5 μl of each culture was spotted onto Luriaagar and incubated for 16 h at 37° C. to enumerate cfu. Data representresults from three independent experiments.

Referring now to FIG. 17A-FIG. 17F plots of bactericidal activity ofpolymyxin B against UPEC in spiked urine samples are shown. UPEC cellswere added to human urine samples and exposed to different polymyxin Bconcentrations. At indicated times, 5 μl of each suspension wasinoculated onto Luria agar. After incubation at 37° C. for 16 h,colonies were enumerated and recorded as cfu/ml. The bars with verticalstriping represent the initial cfu/ml of UPEC cells in urine, and barswith horizontal striping represent cfu/ml of UPEC cells upon exposure toclinically-relevant concentrations of polymyxin B (antibiotics addedimmediately after the baseline measurement) in urine. After addition ofa high, bactericidal concentration of polymyxin B (0.5 mg/ml added afterthe 90-min measurement) and an additional 15-min incubation, no colonieswere detected on Luria agar. These results indicated that the polymyxinB MBC against UPEC cells spiked in human urine was of 2 μg/ml.

Referring now to FIG. 18A, motion of a living bacterial cell over timeis shown: FIG. 18A shows displacement of an alive bacterial cell over 5s in 1×PBS. The position of the bacterial cell at 0 s (marked by *) andvarious other time points (marked by o) is shown.

Referring now to FIG. 19A and FIG. 19B, motion of a dead bacterial cellover time. (a) Displacement of a dead bacterial cell over 5 s in 1×PBS.The position of the bacterial cell at 0 s (marked by *) and variousother time points (marked by o) is shown. (b) Distance of a deadbacterium after 5 mins in 0.5 mg/ml polymyxinB over triplicate 20 srecordings. At 20 s, the distance from triplicates is averaged to obtainmotion of the cell at 5 mins in 0.5 mg/ml polymyxin B.

Referring now to FIG. 20A-FIG. 20C, motion changes of loosely andcompletely tethered bacterial cells (a) D_(AVG) of loosely tetheredbacterial cells shows a significant decrease after addition of 0.5 mg/mlpolymyxin B. (b) D_(AVG) of completely tethered bacterial cells show nochange after addition of 0.5 mg/ml polymyxin B. (c) A completelytethered alive bacterium in 1×PBS shows no changes in distance after 10mins in 0.5 mg/ml polymyxin B.

Certain exemplary embodiments of the invention have been describedherein in considerable detail in order to comply with the PatentStatutes and to provide those skilled in the art with the informationneeded to apply the novel principles of the present invention, and toconstruct and use such exemplary and specialized components as arerequired. However, it is to be understood that the invention may becarried out by different equipment, and devices, and that variousmodifications, both as to the equipment details and operatingprocedures, may be accomplished without departing from the true spiritand scope of the present invention.

As another example, measuring sub-μm motion changes of loosely boundmicroorganisms after they interact with antibiotics correspond to ameasure of their susceptibility to antibiotics, leading to a new andrapid way to perform Antibiotic Susceptibility Test (AST), a criticaldiagnostic test currently. Antibiotic susceptibility information forsingle bacterial cells (single cell antibiotic susceptibility) isprovided using the above invention, which is not possible using currentassays which measure bulk populations. Single cell susceptibilityanalysis can also be used for clinical cases of polymicrobial infectionsand to identify resistant cells in a population, which is not possibleusing current bulk assays.

REFERENCES

The teachings of the following publications are incorporated herein intheir entirety by this reference.

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What is claimed is:
 1. A method for measuring the sub-micron motion ofsurface-tethered bacterial cells comprising: adding different doses ofat least one antibiotic to different ones of a plurality of wells in amulti-well glass slide, each well having a surface; adding portions of abiological sample with live bacteria to the plurality of wells,incubating bacterial cells from the live bacteria tethering a pluralityof bacterial cells onto one of the surfaces to create tethered bacterialcells; operating a light microscopy apparatus to image each of theplurality of wells including imaging the tethered bacterial cells;operating a camera to obtain images of the tethered bacterial cells fromthe light microscopy apparatus; tracking the sub-micron motion of imagesof the tethered bacterial cells; analyzing bacterial motion of tetheredcells in different wells at the different doses; operating a processorto apply an image analysis process to the images to quantitate themotions of the tethered bacterial cells so as to detect changes in themotions of the tethered cells; storing the motion changes in memory;operating a processor to perform statistical analysis on a population ofcells for each antibiotic dose to generate an antibiotic dose curveproportional to the motion changes; and operating an imaging device anda processor for measuring the X and Y displacement of each tethered cellcenter by subtracting the X and Y coordinates of an individual cell fromthe cell's average position over the length of the image sequences andcalculating a distance moved by the tethered cell center by using theformula distance=√{square root over (σx²+σy²)}, where σx and σyrepresents the standard deviation of X and Y displacement, respectively.2. The method of claim 1 wherein the surface is a glass surface furthercomprising treating the glass surface with linkers selected from thegroup consisting of APTES, antibodies, poly-I-lysine and tetheringmolecules.
 3. The method of claim 1 wherein tethering bacterial cellscomprises attaching tethering molecules to the surface, where thetethering molecules are selected from the group consisting ofcell-adhesion promoting substances, poly-lysine, and agar matrix.
 4. Themethod of claim 1 wherein the act of operating a processor to performstatistical analysis on a population of cells for each antibiotic doseto generate an antibiotic dose curve proportional to the motion changesfurther comprises: determining, using an imaging device and a processor,a distance range of bacterial cell motion; determining, using aprocessor, whether a distance range of bacterial cell motion is between50 nm and 65 nm; determining, using an imaging device and a processor, adose of antibiotic used to limit the distance range of bacterial cellmotion between 50 nm and 65 nm; and reporting the dose as the minimumbactericidal concentration of antibiotic for the antibiotic.
 5. Themethod of claim 1 further comprising operating a gravity-basedmultichannel drug perfusion system to deliver medium and buffers to theplurality of wells.
 6. The method of claim 1 further comprising applyingglass coverslips coated with chromium and to the multi-well glass slideas a sensor surface.
 7. A method for rapid antibiotic susceptibilitytesting by tracking sub-micron scale motion of single bacterial cellscomprising: adding different doses of at least one antibiotic todifferent ones of a plurality of wells in a multi-well glass slide, eachwell having a surface; adding portions of a biological sample with livebacteria to the plurality of wells, incubating bacterial cells from thelive bacteria to tether a plurality of bacterial cells onto one of thesurfaces to create tethered bacterial cells; operating a lightmicroscopy apparatus to image each of the plurality of wells includingimaging the tethered bacterial cells; operating a camera to obtainimages of the tethered bacterial cells from the light microscopyapparatus; tracking the sub-micron motion of images of the tetheredbacterial cells; analyzing bacterial motion of tethered cells indifferent wells at the different doses; operating a processor to applyan image analysis process to the images to quantitate the motions of thetethered bacterial cells to detect changes in the motions of thetethered cells; storing the motion changes in memory; operating aprocessor to perform statistical analysis on a population of cells foreach antibiotic dose to generate an antibiotic dose curve proportionalto the motion changes, wherein the antibiotic dose curve plots dataincluding a decrease in movement over time indicating a proportionaleffectiveness of an antibiotic applied to a well; and operating animaging device and a processor for measuring the X and Y displacement ofeach tethered cell center by subtracting the X and Y coordinates of anindividual cell from the cell's average position over the length of theimage sequences and calculating a distance moved by the tethered cellcenter by using the formula distance=σx²+σy², where σx and σy representsthe standard deviation of X and Y displacement, respectively.
 8. Themethod of claim 7 further comprising creating the tethered bacterialcells by incubating the cells with APTES in Ethanol, incubated on thesurface for up to 15 seconds.
 9. The method of claim 7 wherein tetheringthe antibody comprises incubating 30 ug/ml of antibody solution for upto 30 mins on a PEG/PEG-000H surface.
 10. The method of claim 7 furthercomprising removing unattached bacterial cells prior to the act ofoperating a light microscopy apparatus to image each of the plurality ofwells.
 11. The method of claim 7 further comprising operating agravity-based multichannel drug perfusion system to deliver medium andbuffers to the plurality of wells.
 12. The method of claim 7 furthercomprising applying glass coverslips coated with chromium and gold tothe multi-well glass slide as a sensor surface.
 13. A method for rapidantibiotic susceptibility testing by tracking sub-micron scale motion ofsingle bacterial cells comprising: adding different doses of at leastone antibiotic to different ones of a plurality of wells in a multi-wellglass slide, each well having a surface; adding portions of a biologicalsample with live bacteria to the plurality of wells, incubatingbacterial cells from the live bacteria to tether a plurality ofbacterial cells onto one of the surfaces as tethered bacterial cells,where the tethered bacterial cells are incubated with APTES in Ethanolon the surface for up to 1 5 seconds; operating a light microscopyapparatus to image each of the plurality of wells including imaging thetethered bacterial cells; operating a camera to obtain images of thetethered bacterial cells from the light microscopy apparatus; trackingthe sub-micron motion of images of the tethered bacterial cells;analyzing bacterial motion of tethered cells in different wells at thedifferent doses; operating a processor to apply an image analysisprocess to the images to quantitate the motions of the tetheredbacterial cells to detect changes in the motions of the tethered cells;storing the motion changes in memory; operating a processor to performstatistical analysis on a population of cells for each antibiotic doseto generate an antibiotic dose curve proportional to the motion changes,wherein the antibiotic dose curve plots data including a decrease inmovement over time indicating a proportional effectiveness of anantibiotic applied to a well; and operating an imaging device and aprocessor for measuring the X and Y displacement of each tethered cellcenter by subtracting the X and Y coordinates of an individual cell fromthe cell's average position over the length of the image sequences andcalculating a distance moved by the tethered cell center by using theformula distance=√{square root over (σx²+σy²)}, where ax and ayrepresents the standard deviation of X and Y displacement, respectively.14. The method of claim 13 wherein tethering the antibody comprisesincubating 36 ug/ml of antibody solution for up to 30 mins on aPEG/PEG-000H surface.
 15. The method of claim 13 further comprisingremoving unattached bacterial cells prior to the act of operating alight microscopy apparatus to image each of the plurality of wells. 16.The method of claim 13 further comprising operating a gravity-basedmultichannel drug perfusion method to deliver medium and buffers to theplurality of wells.
 17. The method of claim 13 further comprisingapplying glass coverslips coated with chromium and gold to themulti-well glass slide as a sensor surface.
 18. The method of claim 1wherein the live bacteria are selected from the group consisting ofEscherichia coli and uropathogenic E. coli.
 19. The method of claim 7wherein the live bacteria are selected from the group consisting ofEscherichia coli and uropathogenic E. coli.
 20. The method of claim 13wherein the live bacteria are selected from the group consisting ofEscherichia coli and uropathogenic E. coli.